2,3-Benzodiazepines
Certain 2,3-benzodiazepines have been explored extensively for their potent CNS modulating activity. Compounds such as tofisopam (Grandaxin®), girisopam, and norisopam (structures shown below with the atom numbering system indicated for tofisopam) have demonstrated substantial anxiolytic and antipsychotic activity.

Tofisopam has been shown in humans to have an activity profile that is significantly different from that of widely used 1,4-benzodiazepine (BZ) anxiolytics such as diazepam (Valium®) and chlordiazepepoxide (Librium®). The 1,4-benzodiazepines, in addition to having sedative-hypnotic activity, also possess muscle relaxant and anticonvulsant properties that, though therapeutically useful in some disease states, are nonetheless potentially untoward side effects. Thus, the 1,4-benzodiazepines, though safe when administered alone, may be dangerous in combination with other CNS drugs including alcohol.
Tofisopam, in contrast, is a non-sedative anxiolytic that has no appreciable sedative, muscle relaxant or anticonvulsant properties. See Horvath et al., Progress in Neurobiology, 60 (2000), 309–342; the entire disclosure of which is incorporated herein by reference. In clinical studies, tofisopam improved rather than impaired psychomotor performance and showed no interaction with ethanol (Id.). These observations comport with data that show that tofisopam does not interact with central benzodiazepine (BZ) receptors and binds only weakly to peripheral BZ receptors. Additional studies have shown that tofisopam enhances mitogen-induced lymphocyte proliferation and IL-2 production in vitro. (Id)
Other 2,3-benzodiazepines, though structurally similar to tofisopam, have been investigated and shown to have varying activity profiles. For example, GYKI-52466 and GYKI-53655 (structures shown below) act as noncompetitive glutamate antagonists at the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) site, and have demonstrated neuroprotective, muscle relaxant and anticonvulsant activity (Id.). Another group of 2,3-benzodiazepines that have been investigated are represented by the compound GYKI-52895, and show activity as selective dopamine uptake inhibitors with potential use in antidepressant and anti-Parkinsonism therapy (Id.).

Tofisopam is a racemic mixture of (R)- and (S)-enantiomers. This is due to the asymmetric carbon, i.e., a carbon with four different groups attached, at the 5-position of the benzodiazepine ring. The molecular structure and conformational properties of tofisopam have been determined by NMR, CD and x-ray crystallography (Visy et al., Chirality 1:271–275 (1989)). The 2,3-diazebine ring exists as two different conformers. The major conformers, (+)R and (−)S have the 5-ethyl group in a quasi-equatorial position, while in the minor conformers, (−)R and (+)S, the 5-ethyl group is positioned quasi-axially. Thus, racemic tofisopam can exist as four molecular species, i.e., two enantiomers, each of which exists as two conformations. The sign of the optical rotation is reversed upon inversion of the diazepine ring from one conformer to the other. In crystal form, tofisopam exists only as the major conformations, with dextrorotatory tofisopam being of the (R) absolute configuration. (Toth et al., J. Heterocyclic Chem., 20:709–713 (1983); Fogassy et al., Bioorganic Heterocycles, Van der Plas, H. C., Ötvös, L, Simongi, M., eds. Budapest Amsterdam: Akademia; Kiado-Elsevier, 229:233 (1984)).
Differential binding of these two conformations of tofisopam has been reported in binding studies with human albumin (Simongi et al. Biochem. Pharm., 32(12), 1917–1920, 1983). The two conformers have also been reported as existing in equilibrium (Zsila et al., Journal of Liquid Chromatography & Related Technologies, 22(5), 713–719, 1999; and references therein).
The optically pure (R)-enantiomer of tofisopam (R)-1-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7,8-dimethoxy-5H-2,3-benzodiazepine) has been isolated and shown to possess the nonsedative anxiolytic activity of the racemic mixture. See U.S. Pat. No. 6,080,736; the entire disclosure of which is incorporated herein by reference.
Neutropenia
Neutrophils, also called polymorphonuclear leukocytes, are the most numerous of the blood cells known as granulocytes. Neutrophils are the largest cell population involved in acute inflammatory response. They are thus an important component of natural immunity, responding quickly to chemotactic stimuli. Neutrophils destroy foreign particles such as bacteria by enveloping and digesting them, a process called phagocytosis. Neutrophils increase in response to bacterial infection. When many neutrophils are needed, they are released from the bone marrow as immature cells, called bands or stab cells.
Neutropenia is a blood disorder wherein the number of neutrophils in the blood is abnormally low as assessed by an Absolute Neutrophil Count (ANC). An ANC is acquired by performing a differential blood cell count in which percentages of cell types are recorded as well as the total number of cells. The differential blood cell count is done by spreading a drop of blood on a microscope slide. The slide is stained and examined under a microscope. One hundred white cells are counted and identified as either neutrophils, bands, lymphocytes, monocytes, eosinophils or basophils. Any a typical or immature cells also are counted. Cells are identified by the shape and appearance of the nucleus, the color of cytoplasm (the background of the cell), and the presence and color of granules. The percentage of each cell type is reported. Some instruments may be used to perform an automated differential.
A neutrophil shortage corresponds to an increased risk of microbial infection. The blood of healthy human adults contains about 2500 to 6000 neutrophils per mm3. In children under the age of six, the count may be lower. Various sources have set the threshold for the diagnosis of neutropenia at different measured neutrophil levels ranging from an ANC of about 2000 neutrophils per mm to about 1500 neutrophils per mm3. See The Merck Manual 17th Ed. 1999, Section 11, Chapter 135, and Wallner, et al., U.S. Pat. No. 6,300,314, Oct. 9, 2001, the entire disclosures of which are incorporated herein by reference. Severe neutropenia is diagnosed when the ANC falls below 500 neutrophils per mm3. The symptoms, of increased risk of infection depend on the severity of the neutropenia and on the duration of the disorder.
Neutropenia treatable by methods of the present invention may be a chronic disorder. Neutropenia as a chronic disorder may be further classified as congenital, cyclical and idiopathic neutropenia. Chronic congenital neutropenia is inherited by a small number of individuals. The most severe form of congenital neutropenia is Kostmann's Syndrome and there are other, milder variations. Symptoms include frequent infections and fevers.
Cyclical neutropenia results from a regulatory defect at the hematopoietic stem cell level that causes oscillations in production of neutrophils as well as other types of blood cells. Individuals with this disorder will have neutrophil counts of about 100 neutrophils per mm3 for three to six days out of every cycle. The neutrophil count ranges from severe to moderate neutropenia levels through most of the cycle.
Chronic idiopathic neutropenia refers to severe chronic neutropenia that does not clearly fall into either of the above classifications. Individuals suffering from chronic idiopathic neutropenia typically acquire the disorder after having normal neutrophil counts earlier in life. It is estimated that neutropenia may occur as a congenital or idiopathic disorder in an estimated frequency of one per 200,000 in the population.
Neutropenia may also occur secondary to another condition such as cancer or Acquired Immunodeficiency Syndrome. Neutropenia may also occur secondary to an event such as a drug therapy. Thus, neutropenia may result from physiological disorders that directly affect the immune system. For example, diminished neutrophil production will result when leukemia, myeloma, lymphoma or a metastatic solid tumor such as, for example, breast or prostate cancer, infiltrate and replace bone marrow. Transient neutropenia is often associated with viral infections. Chronic neutropenia is often associated with immunodeficiency resulting from a viral infection, for example, Acquired Immunodeficiency Syndrome (AIDS) resulting from infection with Human Immunodeficiency Virus (HIV). Autoimmune neutropenia may be associated with circulating antineutrophil antibodies.
A much more common cause is neutropenia as a side effect of drug therapy, particularly cancer chemotherapy, radiation therapy for cancer and bone marrow transplantation associated with cancer therapy. Neutropenia secondary to drug therapy can thus be subdivided into two groups. The first involves immune-mediated neutropenia that may arise from drugs that act as haptens to stimulate antibody formation. Acute hypersensitivity reactions such as those caused by diphenylhydantoin and phenobarbital may last a few days. However, chronic hypersensitivity reactions may last for months or years. See The Merck Manual, 17th Ed., Id.
The second area of drug-induced neutropenia involves the severe neutropenia that predictably occurs after large doses of cytoreductive cancer drugs and which also accompanies ionizing radiation therapy. These cytotoxic therapies induce neutropenia because of the proliferative nature of neutrophil precursor cells and the normal rapid turnover rate of circulating neutrophils. See The Merck Manual, 17th Ed., Id. The risk of neutropenia secondary to cancer chemotherapy or radiotherapy depends on the type and stage of the cancer and the type, the dosage and the schedule of cancer treatment. Approximately 1.4 million cancer patients in the US received chemotherapy in 2001. About one half of chemotherapy patients develop neutropenia. At present, less than 10% of chemotherapy patients receive prophylactic treatment to prevent neutropenia.
Therapy that exists currently for raising neutrophil levels consists primarily of filgrastim (Nupogen®) and more recently, pegfilgrastim (Neulasta™), a longer acting derivative of filgrastim. Filgrastim is a recombinant version of a human protein, G-CSF (granulocyte-colony stimulating factor), that selectively stimulates the production of white blood cells. G-CSF is currently the drug of choice for neutropenia. Since both of these drugs are recombinant proteins they are not active orally and must be administered by injection. In addition, protein-based drugs are often subject to rapid metabolism. The elimination half-life of Nupogen® is 3.5 hours and of Neulasta™ is in the range of 15–80 hours.
New agents are needed which are useful in the treatment of neutropenia. In particular, agents are needed that demonstrate biological activity when administered via routes other than injection. Particularly, agents that may be orally active are needed, as they may serve to enhance patient compliance.